MXPA00011580A - Genes encoding epsilon lycopene cyclase and method for producing bicyclic epsilon carotene. - Google Patents

Abstract

The present invention relates to the DNA sequence for eukaryotic genes encoding epsilon cyclase isolated from romaine lettuce as well as vectors containing the same and hosts transformed with said vectors. The present invention provides methods for controlling the ratio of various carotenoids in a host and to the production of novel carotenoid pigments. The present invention also provides a method for treating disease by administering carotenoids obtained from transformed hosts, or by administering a composition containing the transformed hosts.

Description

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GENES THAT CODI FICAN EPSILON LICOP ENO CYCLASA AND M ETHOD FOR PRODUCI R EPSILON CAROTENO BIC1CLICO BACKGROUND OF THE INVENTION FIELD OF THE INVENTION The present invention describes the DNA sequence for eukaryotic genes encoding lycopene cyclase as well as vectors containing them and hosts transformed with these vectors The present invention also provides a method for increasing the aquatic mutation of carotenoids. and Production of Novel and Rare Carotenoids The present invention provides methods for controlling the proportion of various carotenoids in a host.Additionally, the present invention provides a method for protecting eukaryotic genes encoding carotenoid biosynthesis and metabolism enzymes. it also provides transgenic plants having therapeutic properties, methods for preparing a therapeutic composition, and methods for treating diseases by administering therapeutic therapeutics and compositions.

DISCUSSION OF THE BACKGROUND Carotenoid pigments with cyclic end groups are essential components of the photographic apparatus. ntético in oxygenic photosynthetic organisms (v. g., cyanobacteria, algae and plants; Goodwin, 1 980). The symmetrical bicyclic carotenoid yellow pigment β-carotene (or, in rare cases, the asymmetric bicyclic α-carotene) is intimately associated with the photosynthetic reaction centers and plays a vital role in the protection against potentially lethal photooxidative damage (Koyama, 1991) . The ß-carotene and other carotenoids derived from this or a-carotene also serve as light harvesting pigments (Stefermann-Harms, 1987), are involved in the thermal dissipation of excess light energy captured by the harvest antenna. Light (Demming-Adams &Adams, 1992), provide substrate for the biosynthesis of abscisic acid plant growth regulator (Rock &Zeevaart, 1 991; Parry &Horgan, 1991), and are precursors of vitamin A in diets of humans and animals (Krynsky, 1987). Plants also exploit the carotepoides as coloring agents in flowers and fruits to attract pollinators and seed dispersing agents (Goodwin, 1 980). The color provided by carotenoids is also of agronomic value in a number of important crops. Carotenoids are normally harvested from plants for use as pigments in food and feed. In the higher plant carinoenoids, two types of cyclic end groups are commonly found, these are referred to as the β and e cyclic end groups (Figure 2: the acyclic end group is referred to as the "terminal group" or "psi"). These cyclic terminate groups differ only in the position of the double bind in the an ifis. Carotenoids with two ß rings are ubiquitous, and those with one ß and one ring are common, but carotenoids with two rings are found in significant amounts in relatively few plants. The β-carotene (Figure 1) has two β-terminal groups and is a symmetric compound that is the precursor to a number of other important carotenoids of the plant such as zeaxanthin and violaxanthin (Figure 1). Carotenoid enzymes have previously been isolated from a variety of sources including bacteria (Armstrong et al., 1989, Mol. Gen. Genet 21 6, 254-268; Misawa et al., 1990, J. Bacteriol., 172, 6704. -12), fungi (Schmid hauser et al., 1990, Mol.Cell. Biol., 10, 5064-70), cyanobacteria (Chamovitz et al., 1990, Z. Naturforsch, 45c, 482-86) and higher plants (Bartley and collaborators, Proc. Natl. Acad. Sci., USA 88, 6532-36; Martinez-Ferez &Vioque, 1992, Plant Mol. Biol., 18, 981 -83). Many of the isolated enzymes show a great diversity in functional and inhibitory properties between sources. For example, phytoen desaturases from Synechococcus and higher plants carry a two step desaturation to give? -carotene as a reaction product; while the same Erwinia enzyme introduces four double bonds that form lycopene. Similarly, the amino acid sequences are very low for bacterial enzymes vs. those of plant. Therefore, even with a gene in hand from a source, it is difficult to screen a gene with similar function in another source. In particular, the sequence similarity between bacterial / fungal and cyanobacterial / plant genes is very low.

The difficulties in isolating related genes is exemplified by recent efforts to isolate the enzyme that catalyzes the formation of β-carotene from the precursor acyclic lycopene. Although this enzyme has been isolated in a bacterium, prior to the invention described in Serial No. 08/142, EU 195 (which is hereby incorporated by reference in its entirety), it had not been isolated from any organism. photosynthetic neither had the corresponding genes been identified and sequenced or the co-factor requirements established. The isolation and characterization of the enzyme that catalyzes the formation of β-carotene in the cyanobacterium Synechococcus PCC7942 was described by Cunningham et al. In 1993 and 1994. Arabidopsis β-cyclase adds two rings to the lycopene. -bicyclic carotene, but the relative e-cyclase of Arabidopsis, (which has 36% identity for the predicted amino acid sequences) adds only a single ring to form the monocyclic d-carotene (Cunningham et al., 1996, Plant Cell 8 : 161 3-1626; EU Application No. 08/624, 125 filed March 29, 1996, which is incorporated herein by reference in its entirety). These differences in function provide a simple mechanism for adjusting the proportions of ß, ß- and ß, e-carotenoids while at the same time avoiding the formation of carotenoids with two or three years. In view of the aforementioned deficiencies with methods of the prior art to produce carotenoids with two episid rings, it is clear that there is a need in the art for such methods.

BRIEF DESCRIPTION OF THE INVENTION In short, a first objective of this invention is to provide isolated eukaryotic genes encoding enzymes which encode lycopene cyclases that form bicyclic epsilon-carotene. A second objective of the present invention is to provide vectors containing said genes. A third objective of the present invention is to provide transformed hosts with said vectors. A further object is to provide a method for producing an epsilon cyclase of lycopene using the transformed host. A still further objective is to provide the lycopene cyclase thus produced. Another objective of the present invention is to provide hosts that accumulate novel or rare carotenoids or that overexpress known carotenoids. Still another object of the invention is to provide a method for producing novel or rare carotenoids. Another objective of this invention is to ensure the expression of eukaryotic genes related to carotenoids in a recombinant prokaryotic host. A further object of the invention is a method for preparing a therapeutic composition comprising either the host cell expressing lycopene epsilon cyclase or the isolated carotenoids produced by the cell containing the lycopene epsilon cyclase. Another object of the invention is to provide a method for treating diseases by providing a patient in need thereof, an amount of the rare carotenoids in an amount sufficient to treat the disease. These and other objects of the present invention have been made by the present inventors as described below.

BRIEF DESCRIPTION OF THE DRAWINGS A more complete appreciation of the invention and many of the advantages thereof will be readily obtained as it is better understood by reference to the following detailed description when taken in consideration with reference to the accompanying drawings, wherein : Figure 1 represents possible routes of synthesis of cyclic carotenoids and some xanthophylls of plants and common algae (oxycarotenol) of lycopene. The activities of the e-cyclase enzyme of lettuce are indicated by marked arrows labeled with e. The reaction leading to e-carotene from d-carotene is not catalyzed by the lycopene e-cyclase from Ara bidopsis (Cunningham 1996, Serial No. 08/624, 125 from E.U.) or other known e-cyclases. Therefore, the formation of e-carteno and carotenoids derived therefrom has now become possible with the lycopene e-cyclase of lettuce described herein. Arrows labeled with β indicate synthesis reactions by β-cyclase.

Figure 2 represents the terminal carotene groups that are commonly found in plants. Figure 3 is a DNA sequence of the cDNA (SEQ ID NO: 1) of romaine lettuce encoding the lycopene epsilon cyclase. Figure 4 is the predicted amino acid sequence of the epsilon cyclase (SEQ I D NO: 2) of lycopene from romaine lettuce. Figure 5 is a comparison between the predicted amino acid sequences of romaine lettuce (from clone DY4, SEQ ID NO: 2) and lycopene cyclase from Arabidopsis (from clone y2, SEQ ID NO: 3). Figure 6 shows the nucleotide and amino acid sequences of the e-cyclase # 3 of Adonis palaestina, which also forms bicyclic epsilon carotene (SEQ ID NO: 4 and 5). Figure 7 shows a sequence comparison of Adonis palaestina e-cyclase # 3 (SEQ ID NO: 5) compared to Adonis palaestina e-cyclase # 5 (SEQ ID NO: 6), the last of the four add only a single epsilon ring to lycopene. Five amino acid differences are noted, which may be targets for site-directed mutagenesis to form the lycopene e-cyclase which adds two rings to lycopene.

DETAILED DESCRIPTION Romaine lettuce is one of the rare plant species that produces an abundance of a carotenoid with two epsilon rings (lactucaxanthin). The present inventors have isolated a gene encoding epsilon cyclase from this plant, and have found that it is similar in sequence to that of Arabidopsis (approximately 65% identity). However, the lettuce enzyme efficiently adds two epsilon ai lycopene rings to form the bicyclic epsilon-carotene. The present invention also relates to methods for transforming known carotenoids into novel or rare products. That is, the e-carotene (see Figure 1) and? -carotene streams can be isolated only in minor amounts. As described below, the enzymes of the invention can be produced and used to transform lycopene into bicyclic e-carotene. With such a product at hand, bulk biosynthesis of other carotenoids derived from the bicyclic epsilon carotene is possible. Eukaryotic genes in the trajectory of carotenoid biosynthesis differ from their prokaryotic counterparts in their 5 'region. As used herein, the 5 'region is the region of eukaryotic DNA that precedes the initiation codon of the counterpart gene in prokaryotic DNA. That is, when consented areas of eukaryotic and prokaryotic genes align, eukaryotic genes contain additional coding sequences upstream of the prokaryotic codon and initiation. The invention also relates to genes encoding epsilon c? Class of lycopene which are truncated in the 5 'region of the gene. Preferably, such truncated genes are truncated to codons within 0-50, preferably 0-25 of the 5 'initiation codon of their prokaryotic counterparts as determined by alignment maps.

In addition to the novel enzymes produced by truncating the 5 'region of known enzymes, novel enzymes can be formed that can participate in the formation of novel carotenoids by replacing portions of a gene with an analogous sequence of a structurally related gene. Information to add two epsilon rings can be found in the 3 'half of the romaine lettuce gene. Thus, an example of such a hybrid gene construct would include the first half of the romaine lettuce gene in coation with the second half (3 ') of another plant cyclase gene, such as the potato gene or by mutagenesis randomization. directed on site of a mono-cyclase.

Vectors Genes encoding carotenoid enzymes as described above, when cloned into a suitable expression vector, can be used to overexpress these enzymes in a plant expression system or to inhibit the expression of these vectors. The production or biochemical activity of the gene product epsilon-cyclase and cDNAs can be reduced or inhibited by a * number of different approaches available to those skilled in the art [including, but not limited to such methodologies or approaches as antisense (eg, Gray et al. (1992) Plant Mol. Biol. 19: 69-87), ribozymes (eg, Wegener et al. (1994) Mol. Gen. Genet 245: 465-470), co-suppression (eg, Fray and Grierson (1993) Plant Mol. Biol. 22: 589-602), gene disruption (v. G., Schaefer et al. (1 997) Plant J.11: 195-1206), intracellular antibodies (see g., Rondón and Marasco (1997) Ann.Rev.Microbion 51: 257-283 or any other approaches based on the knowledge or availability of the gene, cDNA , or polypeptide and / or sequences thereof] whereby the accumulation of carotenoids with psilon rings and compounds derived therefrom is reduced.For example, a vector containing the gene encoding e-cyclase can be used for increase the amount of bicyclic épsiion-carotene in an organism and with this alter the nutritional value, pharmacological value and visual appearance of the organism Furthermore, the transformed organism can be used in the formulation of therapeutic agents, for example in the treatment of cancer (Mayne et al. (1996) FASEB J. 10: 690-701; Tsushima and collaborators(1995) Biol. Pharm. Bull. 18: 227-233, which are incorporated into the present by reference in their totalities). In a preferred embodiment, the vectors of the present invention contain a DNA encoding a eukaryotic IPP isomerase upstream of a DNA encoding a second eukaryotic carotenoid enzyme. The inventors have discovered that the inclusion of an isomerase I PP gene increases the substrate substrate for the carotenoid path; whereby the production of final carotenoid products is increased. This is apparent from the much more intense pigmentation in colonies of E. coli that accumulate carotenoids which also contain one of the aforementioned isoprase I PP genes when compared to colonies lacking this additional IPP isomerase gene. Similarly, a vector comprising an IPP isomerase gene can be used to increase the production of secondary metabolites of dimethylallyl pyrophosphate (such as isoprenoids, steroids, carotenoids, etc.). Alternatively, an anti-sense strand of one of the above genes can be inserted into a vector. For example, the e-cyclase gene can be inserted into a vector and incorporated into the genomic DNA of a host, thereby inhibiting the synthesis of e, ß carotenoids (lutein and a-carotene) and increasing the synthesis of epsilon bicyclic carotenoids. Suitable vectors according to the present invention comprise a eukaryotic gene that encodes an enzyme involved in biosynthesis or carotenoid metabolism and a suitable promoter so that the host can be constructed using techniques well known in the art (eg, Sambrook et al. , Molecular Cloning A Laboratorv Manual. Cold Spring Harbor, NY, 1989). Vectors suitable for eukaryotic expression in plants are described in Plant J. de Frey et al., (1995) 8 (5): 693 and Misawa et al., 1994; incorporated herein by reference in their totalities. Vectors suitable for prokaryotic expression include pACYC1 84, pUC 1 19, and pBR322 (available from New England BioLabs, Bevery, MA), pTrcHis (Invitrogen), Bluescript SK (Stratagene) and pET28 (Novagen) and its derivatives. The vectors of the present invention may additionally contain regulatory elements such as promoters, selectable marker repressors such as antibiotic resistance genes, etc.

Hosts Host systems according to the present invention can comprise any organism that already produces carotenoids or which has been genetically modified to produce carotenoids. Organisms that already produce carotenoids include plants, algae, some yeasts, fungi, and cyanobacteria and other photosynthetic bacteria. The transformation of these convective hosts according to the present invention can be done using standard techniques such as. those described in Misawa et al., (1990) supra; Hundle et al., (1993) supra; Hundle et al., (1991) supra; Misawa et al. (1991) supra; Sandmann et al., Supra; and Schnurr and collaborators, his pra; all incorporated herein by reference in their totalities. E. coli is an example of a type of bacterium that is suitable as a host for expression of the present enzymes (Cun ningham et al. (1996) The Plant Cell 8: 1613-1626, which is incorporated herein by reference in its entirety). A vector is used to construct plasmids containing genes encoding the enzymes of the invention, said vector allows them to coexist in E. coli with cloning vectors containing the most common ColEl origin of duplication. The addition of cyclic epsilon terminal groups for pink colored lycopene will result in products that are yellow or orange-yellow in color. Therefore, the functioning of the lycopene cyclase cyclase of the invention can be detected by a change in the color of E. coli cultures that accumulate lycopene. Such assays are complementation tests qualified by color. Alternatively, transgenic organisms can be constructed which include the DNA sequences of the present invention (Bird et al., 1991; Bramley et al., 1992; Misawa et al., 1994a; Misawa et al., 1994b; Cunningham et al., 1993, all of which are incorporated by reference to the present in their totalities). The incorporation of these sequences can make it possible to control the biosynthesis, content, or composition of carotenoid in the host cell. These transgenic systems can be constructed to incorporate sequences that allow the overexpression of the carotenoid genes of the present invention. Transgenic systems can also be constructed containing antisense expression of the DNA sequences of the present invention. Such antisense expression would result in the accumulation of the substrates of the enzyme encoded by the sense strand. Suitable transgenic hosts include lettuce, the natural host, but also other plants such as calendula, tomato, pepper, banana, potato and the like. Essentially any plant is adequate to express the enzyme present, but preferred plants are those that already produce high levels of carotenoids, and those that are normally ingested as food or used as a source of carotenoid pigments. In particular, fruit-bearing plants can be manipulated in such a manner as to provide specific expression of tissue in fruits. Calendula is a particularly preferred host, because it can be used as a "bioreactor" for bulk production of carotenoids, and is actually growing on the commercial piano as a source of carotenoids for chicken feeding. For the expression in calendula, a promoter which is "specific for flowers" can be used. Another preferred transgenic plant is tomato, because this plant already produces high levels of icopene. In fact, it has been reported that there is a correlation between the consumption of tomatoes and the decrease in incidence of colon cancer (mayne, supra).

A Method for Selecting Eukaryotic Genes Encoding Enzymes Involved in Carotenoid Biosynthesis The method of the present invention comprises transforming a prokaryotic host with a DNA that can contain a biosynthetic gene of carotenoid and eukaryotic or prokaryotic; cultivate said transformed host to obtain colonies; and selecting colonies exhibiting a color different from that of the colonies of the untransformed host. Suitable hosts include E. coli, cyanobacteria such as Synechococcus and SynechoCistis, algae and plant cells. E. coli is preferred. In a preferred embodiment, the above "color complementation test" can be improved using mutants that are either (1) deficient in at least one carotenoid biosynthetic gene or (2) overexpress at least one biosynthetic carotenoid gene. In any case, such mutants will accumulate carotenoid precursors. The genomic and prokaryotic and eukaryotic cDNA literatures can be scrutinized in total for the presence of genes for biosynthesis, metabolism and degradation of carotenoids. The preferred organisms to be screened include photosynthetic organisms, humans and animals. E. coli can be transformed with these eukaryotic cDNA files using conventional methods such as those described in Sambrook et al., 1989 and in accordance with the protocols described by sellers of cloning vectors. For example, cDNA files in bacteriophage vectors such as lambdaZAP (Stratagene) or lambdaZI PLOX (Gibco BRL) can be excised en masse and used to transform E. coli. Suitable vectors include pACYC1 84, pUC1 1 9, pB R322 (available from New England BioLabs, Bevery, MA). The pACYC is preferred. Transformed E. coli can be grown using conventional techniques. The culture broth preferably contains antibiotics to select and maintain plasmids. Suitable antibiotics include penicillin, ampicillin, chloramphenicol, etc. The culture is typically conducted at 5-50 ° C, preferably at ambient temperature (1 6-28 ° C), for 12 hours to 7 days. The cultures are made in plates and the plates are visually screened for colonies with a different color than colonies of the host E. coli transformed with an empty vector. For example, the £. coli transformed with the plasmid, pAC-BETA (described below), produces yellow colonies that accumulate β-carotene. After transformation with a cDNA file, colonies containing a tone different from those formed by E. co /// pAC-BETA would be expected to contain enzymes that modify the structure or degree of expression of beta-carotene. Similar standards that overexpress previous products in carotenoid biosynthesis, such as lycopene, β-carotene, etc., can be manipulated. Having generally described this invention, further understanding can be obtained by reference to certain specific examples which are provided herein for purposes of illustration only and are not intended to be limiting unless otherwise specified.

EJ EMPLO Isolation of lycopene epsilon cyclase Lycopene epsilon cyclase was isolated from a romaine lettuce file obtained from Dr. Harry Y. Yamamoto (University of Hawaii, Honolulu) essentially as described in Cunningham et al., 1996, supra, and Bugos and Yamamoto (1996) Proc. Natl. Acad. Sci. USA 93: 6320-6325, both incorporated herein by reference in their entireties. Functional genes were identified by the color complementation test.

Pigment Analysis A single colony was used to inoculate 50 ml of LB containing ampicillin and chloramphenicol in a 250 ml bottle. The cultures were incubated at 28 ° C for 36 hours with gentle agitation, and then harvested at 5000 rpm in an SS-34 rotor. The cells were washed once with distilled H20 and resuspended with 0.5 ml of water. The extraction and HPLC procedures were essentially as previously described (Cunningham et al., 1994).

Organisms and Growth Conditions Strains of E. coli TOP 1 0 and TOP 10 F '(obtained from Invitrogen Corporation, San Diego, CA) and XL1-Blue (Stratagene) were grown in Luria-Bertani medium (LB). ) (Sambrook et al., 1989) at 37 ° C in the dark on a platform agitator at 225 cycles per minute.

The components of the medium were Difco (extract of yeast and tryptone) or Sigma (NaCl). Ampicillin at 50 μg / mL and / or chloramphenicol at 50 μg / mL (both from United States Biochemical Corporation), as appropriate, for selection and maintenance of plasmids. Mass Suppression and Selection by Color Complementation of Lech uga Romana cDNA file. A roma lettuce cDNA file was obtained in lambda ZAPII (Burgos &Yamamoto) from Henry Yamamoto, as noted above. An aliquot of each file was treated to cause mass suppression of the cDNAs and thereby produce a phagemid file according to the instructions provided by the provider of the cloning vector (Stratagene) E.coli strain XL1 -Blue was used and the phage of assistance R408). The titer of the suppressed phagemid was determined and the file was introduced into a lycopene accumulation strain of E. coli TOP 1 0 F 'by incubation of the phagemid with the E. coli cells for 1 5 minutes at 37 ° C. they grew overnight at 30 ° C in LB medium supplemented with 2% maltose (w / v) and 10 mM MgSO (final concentration), and were harvested in 1.5 ml micro-tube tubes in a 3-place placement. an Eppendorf microfuge (541 5C) for ten minutes. The pellet was resuspended in 10 mM MgSO4 to a volume equal to one half of the initial culture volume. The transformants were disseminated in large Petri dishes with LB agar (150 mm diameter) containing antibiotics to provide for the selection of cDNA clones (ampicillin) and maintenance of pAC-LYC (chloramphenicol). Approximately 10,000 colony forming units were scattered in each plate. The Petri dishes were incubated at room temperature for two to seven days to allow maximum color development. The plates were visually monitored with the aid of a 3x illuminated amplifier and a low-power stage dissection microscope for the rare pale pink to yellow to bright yellow colonies that could be observed in the pink colonies background. A colony color from yellow to pinkish-yellow was taken as presumptive evidence of a cyclization activity. These yellow colonies were harvested with sterile sticks and used to inoculate 3 ml of LB medium in culture tubes with growth overnight at 37 ° C and shaking at 225 cycles / minute. The cultures were divided into two aliquots in microfuge tubes and harvested by centrifugation in a placement of 5 in an Eppendorf 541 5C microfuge. After discarding the liquid, a granule was frozen for further purification of plasmid DNA. To the second granule, 1.5 ml of EtOH was added, and the granule was resuspended by mixing with a vortex, and the extraction was allowed to proceed in the dark for 1 to 30 minutes with occasional remixing. The insoluble materials were granulated by centrifugation at maximum speed for 10 minutes in a microfuge. The absorption spectrum of supernatant fluids was recorded from 350-550 nm with a lambda six Pekin Elmer spectrophotometer.

Analysis of isolated clones Eight of the yellow colonies contained e-carotene indicating that a single gene product catalyses both cyclizations required to form two e-carotene end groups from the lycopene symmetric precursor. The availability of the romaine lettuce gene encoding the cyclase allows targeted manipulation of plant and algae species for modification of carotenoid content and composition. Through the inactivation of the cyclase, either at the gene level by gene knockout or by insertion inactivation or by reducing the amount of enzyme formed (by such antisense technology), one can increase the formation of ß-carotene and other pigments derived from it. Since vitamin A is derived only from carotenoids with β-terminal groups, an increase in the production of β-carotene vs. a-carotene can increase the nutritional value of crop plants. The reduction of carotenoids with terminal groups can also be of value when modifying the color properties of specific plants and tissues of these plants. Alternatively, when the production of α-carotene is desired, or pigments such as lutein that are derived from α-carotene, either by color properties, nutritional value or other reason, one may overexpress the cyclase or express it in specific tissues. . Wherever the agronomic value of culture is related to the pigmentation provided to carotenoid pigments, the targeted manipulation of the expression of the cyclase gene and / or production of the enzyme may be of commercial value. The predicted amino acid sequence of the enzyme (S EQ I D NO: 2) of the cyclase from the Roman leech was determined. A comparison of the amino acid sequences of the cyclase enzymes of Arabidopsis thaliana and romaine lettuce (Figure 5) as predicted by the DNA sequence of the respective genes (Figure 3 for the cyclase cDNA sequence), indicates that these two enzymes have many regions of sequence similarity, but they are only about 65% overall identical at the amino acid level.

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LIQUIDITY OF SEQUENCES < 110 > CUNNINGHAM JR., FRANCIS X. SUN ZAIREN < 120 > GENES THAT CODE ESPSILON CICL? SA UCOPENO AND METHOD TO PRODUCE EPSILON BICYCLIC CAROTENE < 130 > 2747 - 0084 - 27 CIP < 140 > 09 / 084,222 < 141 > 1998 - 05 - 26 < 150 > 08 / 937,155 < 151 > 1997-09-25 < 150 > 08 / 624,125 < 151 > 1996-03-29 < 160 > 6 < 170 > Patentln Ver. 2.0 < 210 > 1 < 211 > 1780 < 212 > DNA < 213 > romaine lettuce < 223 > n is an unspecified nucleotide < 400 > 1 gaaacaaatg acgtgaaagt tcttcaaaat tgaattaatt gtaatcctga aaacttgatt 60 tgtgatagaa gaatcaatgg agtgctttgg agctcgaaac atgacggcaa caatggcggt I20 ttttacgtgc cctagattca cggactgtaa tatcaggcac aaattttcgt tactgaaaca 180 acgaagattt actaatttat cagcatcgtc ttcgttgcgt caaattaagt gcagcgctaa 240 aagcgaccgt tgtgtagtgg ataaacaagg gatttccgta gcagacgaag aagattatgt 300 gaaggccggt ggatcggagc tgttttttgt tcaaatgcag cggactaagt ccatggaaag 360 ctttccgaaa ccagtctaaa agctagcaca gataccaatt ggaaattgca tacttgatct 420 ggttgtaatc ggttgtggcc ctgctggcct tgctcttgct gacgagtcag ccaaactagg 480 gttgaacgtt ggactcattg gccctgatct tccttttaca aacaattatg gtgtttggca 540 ggatgaattt ataggtcttg gacttgaagg atgcattgaa cattcttgga aagatactct 600 tgtatacctt gatgatgctg atcccatccg cataggtcgt gcatatggca gagttcatcg 660 tgatttactt catgaagagt tgttaagaag gtgtgtggaa tcaggtgttt catatctaag 720 gaaagaatca ctccaaagta ctgaagctcc aaatggctat agtctcattg aatgtgaagg 780 attccatgca caatatcacc ggcttgctac tgttgcatca ggggcagctt cagggaaatt 840 tctggagtat gaacttg ggg gtccccgtgt ttgtgtccaa acagcttatg gtatagaggt 900 aacaacccct tgaggttgaa atgatccaga tctaatggtg ttcatggatt atagagactt 1020 catgtctcca acaaaaalat tcttcgagga aacttgttta gcttcaagag aagccatgcc 1,140 aagaacgtac gaagaggaat ggtcgtatat ccccgtaggt ggatcgttac ctaatacaga 1200 acaaaagaat ctcgcatttg gtgctgcagc tagtatggtg caccctgcca cagggtattc 1260 tctttgtcag agttgttcga aagctcctaa ttátgcagca gtcattgcta agattttaag 1320 tctaaagaga acaagatcaa tgatttctct tggaaaatac actaacattt caaaacaagc 1380 atgggaaaca ttgtggccac ttgaaaggaa aagacagcga gcettczttc tattcggact 1440 atcacacatc gtgctaatng atctagagrgg aacacgtaca tttttccgta ctttctttcg 1500 tttgcccaaa tggatgtggt ggggatttt ggggtcttct ttatcttcaa cggatttgat 1560 aatatttgcg ctttatatgt ttgtgatagc acctcacagc ttgacraatgg aactggttag 1620 tctqatccga acatctactt tatggtaaaa caggggcaac gcatatctca ctatatagat 1680 ttagattata taaataatac ccatatcttg catatatata agccttattt atttcttttg 1740 acaacatact tacccccaca atgtttttta cgttaattaz 1780 < 210 > 2 < 211 > 533 < 212 > PRT < 213 > romaine lettuce < 400 > 2 Met Glu Cis Fen Gli Wing Arg Asn Met Tre Wing Tre Met Aia Val Fen 5 10 15 Tre Cis Pro Arg Fen Tre Asp Cis Asn lie Arg His Lis Fen Ser Leu 20 25 30 Leu Lis Gln Arg Fen Tre Asn Leu Being Wing Being Being Leu Arg 35 40 45 Gln He Lis Cis Being Ata Lis Being Asp Arg Cis Val Val Asp Lis Gln 50 55 60 Gii He Ser Va! Wing Asp Glu Giu Asp Tir Val Lis Aia Gli Gli Ser 65 70 75 80 Glu Leu Fen Fen Val Gln Met Gin Arg Tre Lis Ser Met Glu Ser Gln 85 90 95 Ser Us Leu Ser Glu Lis Leu Ala Gin He Pro He Gli Asn Cis I have 100 105 110 Leu Asp Leu Val Val He Gli Cis Gli Pro Aia Gli Leu Ala Leu Wing 115 120 125 Wing Glu Ser Ala Lis Leu Gii Leu Asn Val Gli Leu He Gli Pro Asp 130 135 140 Leu Pro Fen Tre Asn Asn Tir Gli Val Trp Gln Asp Glu Fen He Gli 145 150 155 160 Leu Gli Leu Glu Gli Cis He Glu His Ser Trp Lis Asp Tre Leu Val 165 170 175 Tír Leu Asp Asp Wing Asp Pro Ue Arg Jfe Gli Arg Aía Tir GU Arg 180 185 190 Val His Arg Asp Leu Leu His Glu Glu Leu Leu Arg Arg Cis Val Glu 195 200 205 Ser Gfi Val Ser Tir Leu Ser Ser Lis Val Giu Arg He Tre Glu Wing 210 215 220 Pro Asn Gli Tir Ser Leu He Glu Cis Glu Gli Asn He Tre He Pro 225 230 235 240 Cis Arg Leu Ala Tre Val Ala Ser Gli Ala Aía Ser GU Lis Fep Lea 245 250 250 Glu Tir Giu Leu Gli Gli Pro Arg Val Cis Val Gln Tre Wing Tir Gli 260 265 270 lie Glu Val Glu Val Glu Asn Asn Pro Tir Asp Pro Asp Leu Met Vai 275 280 285 Fen Met Asp Tir Arg Asp Fen Ser Lis His Lis Pro Glu Ser Leu Glu 290 295 300 Aia Lis Tir Pro Tre Fen Leu Tir Vai Met Ala Met Ser Pro Tre Lis 305 310 315 320 He Fen Fen Glu Glu Tre Cis Leu Wing Ser Arg Glu Wing Met Pro Fen 325 330 335 Asn Leu Leu Lis Ser Lis Leu Met Ser Arg Leu Lis Wing Met Gli lie 340 345 350 Arg He Tre Arg Tre Tre Glu Glu Glu Trp Ser Tir He Pro Val Gli 355 360 365 Gli Ser Leu Pro Asn Tre Glu Gln Lis Asn Leu Wing Fen Gli Wing Wing 370 375 380 Wing Ser Met Val His Pro Wing Tre Gii Tir Ser Val Val Arg Ser Leu 385 390 395 400 Ser Glu Ala Pro Asn Tir Ala Ala Ala He Ala Lis Le Le Arg Gln 405 410 415 Asp Gín Ser Lis Gúu Met He Ser Leu Gíi Lis Tir Tre Asn He Ser 420 425 430 Lis Gln Ala Trp Glu Tre Leu Trp Pro Leu Glu Arg Lis Arg Gln Arg 435 440 445 Wing Fen Fen Leu Fen Gli Leu Ser His He Val Leu Met Asp Leu Glu 450 455 460 Gli Tre Arg Tre Fen Fen Arg Tre Fen Fen Arg Leu Pro Lis Trp Met 465 470 475 480 Trp Trp Gli Fen Leu Gli Ser Ser Leu Ser Ter Ter Asp Leu He He 485 490 495 Fen Ala Leu Tir Met Fen Val He Ala Pro His Ser Leu Arg Met Glu 500 505 510 Leu Val Arg His Leu Leu Ser Asp Pro Tre Gli Ala Tre Met Val Lis 515 520 525 Ala Tir Leu Tre lie 530 < 210 > 3 < 211 > 524 < 212 > PRT < 213 > Arabidopsis < 400 > 3 Meí Giu Cis Vai GJ / Ala Arg Asn Fen Ala Aia Met Aia Vai Ser Tre 1 5 10 15 Fen Pro Be Trp Be Cis Arg Arg Lis Fen Pro Val Val Lis Arg Tir 20 25 30 Be Tir Arg Asn He Arg Fen Gli Leu Cis Ser Val Arg Ala Ser Gli 35 40 45 Gli Gli Ser Ser Gli Ser Glu Ser Cis Val Ala Val Arg Glu Asp Fen 50 55 60 Wing Asp Glu Glu Asp Fen Val Lis Wing Gli Gii Ser Glu He Leu Fen 65 70 75 80 Val Gín Met Gln Gfn Asn Lis Asp Met Asp Glu Gln Ser Lis Leu Val 85 90 95 Asp Lis Leu Pro Pro Be He Gli Asp Gli Ala Leu Asp His Val 100 105 1 10 Val He Gli Cis Gli Pro Ala Gli Leu Ala Leu Ala Ala Giu Be Ala 115 120 125 Lis Leu Gli Leu Lis Val Gli Leu He Gli Pro Asp Leu Pro Fen Tre 130 135 140 SO? Asn Tir Gii Vai Trp Giu Asp Giu Fen Asn Asp Leu Gli Leu Gin 145 150 155 160 Lis Cis He Glu His Val Trp Arg Gtu Tre He Val Tir Leu Asp Asp 165 170 175 Asp Lis Pro He Tre He Gli Arg Ala Tir Gli Arg Val Ser Arg Arg 180 185 190 Leu Leu His Glu Glu Leu Leu Arg Arg Cis Vai Glu Ser Gli Val Ser 195 200 205 Tir Leu Ser Ser Lis Val Asp Ser He Tre Glu Wing Ser Asp Gli Leu 210 215 220 Arg Leu Val Wing Cis Asp Asp Asn Asn Val ile Pro Cis Arg Leu Wing 225 230 235 240 Ter Val Ala Ser Gli Ala Ala Ser Gli Lis Leu Leu Gln Tir Glu Val 245 250 255 Gli Gli Pro Arg Val Cis Val Gln Tre Aia Tir Gli Val Glu Val Glu 260 265 270 Val Glu Asn Ser Pro Tir Asp Pro Asp Gln Met Val Fen Met Asp Tir 275 280 285 Arg Asp Tir Tre Asn Glu Lis Val Arg Ser Leu Glu Wing Glu Tir Pro 290 295 300 Tre Fen Leu Tir Wing Met Pro Met Tre Lis Ser Arg Leu Fen Fen Glu 305 310 315 320 Glu Tre Cis Leu Ala Ser Lis Asp Val Met Pro Fen Asp Leu Leu Lis 325 330 335 Tre Lis Leu Met Leu Arg Leu Ser Tre Leu Gli lie Arg He Leu Lis 340 345 350 Tre Tir Glu Glu Glu Trp Ser Tir lie Pro Val Gli Gli Ser Leu Pro 355 360 365 Asn Tre Gtu Gln Lis Asn Leu Ala Fen Gli Ala Ala Ala Ser Met Val 370 375 380 His Pro Wing Tre Gli Tir Val Val Arg Ser Leu Ser Glu Wing Pro 385 390 395 400 Lys Tir Wing Ser Val lie Wing Glu He Leu Arg Glu Glu Tre Tre Lis 405 410 415 Gln He Asn Ser Asn Lie Ser Arg Gln Wing Trp Asp Tre Leu Trp Pro 420 425 430 Pro Glu Arg Lis Arg Gin Arg Fen Fen Fen Leu Fen Gli Leu Aia Leu 435 440 445 He Val Gln Fen Asp Tre Glu Gli He Arg Be Fen Fen Arg Tre Fen 450 455 460 Fen Arg Leu Pro Lis Trp Met Trp Gln Gli Fen LeU Gli Ser Tre Leu 465 470 475 480 Tre Ser Gli Asp Leu Val Leu Fen Wing Leu Tir Met Fen Val He Ser 485 490 495 Pro Asn Asn Leu Arg Lis Gii Leu He Asn His Leu He Ser Asp Pro 500 505 510 Tre Gli Ala Tre Met He Lis Tre Tir Leu Lis Val 515 520 < 210 > 4 < 211 > 1848 < 212 > DNA < 213 > Adonis palaestina < 400 > 4 gagagaaaaa gagtgttata ttaatgttac tgtcgcattc ttgcaacaca tattcagact 60 ccatíttctt gttítctctt caaaacaaca aactaatgtg acggagíatc íagctatgga 120 actacttggt gttcgcaacc tcatctcttc ttgccctgtc tggacttttg gaacaagaaa 180 ccttagtagt tcaaaactag cttataacat acatcgatat ggttcttctt gtagagtaga 240 ttttcaagtg agggctgatg gtggaagcgg gagtagaact tctgttgctt ataaagaggg 300 ttttgtggac gaggaggatt ttatcaaagc tggtggttct gagcttttgt ttgtccaaat 360 gcagcaaaca aagtctatgg agaaacaggc caagctcgcc gataagttgc caccaatacc 420 fffcggagaa tctgtgatgg acttggttgt aataggttgt ggacctgctg gtcíttcact 480 ggctgcagaa gctgctaagc taggcttgaa agttggcett attggtcctg atcttccttt 540 tacaaataat tatggtgtgt gggaagacga gttcaaagat cttggacttg aacgttgtat 600 tggaaggaca cgagcatgct ccatclltata tcttgacaat gatgctcctg tccttattgg 660 tcgtgcatat ggacgagtta gccggeattt gctgcatgaa gagttgctga aaaggtgtgt 720 cgagtcaggt gtatcatatc tgaattctaa agtggaaagg atcactgaag ctggtgatgg 780 ccatagtctt gtagtttgtg aaaacgacat ctttatccct tgcaggcttg ctactgttgc 840 gcttcaggga aícíggagca aacttttgga gtatgaagia ggtggcectc gígtttgtgt 900 ccaaactgct tatggtgtgg aggttgaggt ggagaacaat ccatacgatc ccaacttaat 960 ggtatttatg gactacagag actatatgca acagaaatía cagtgctcgg aagaagaata 1020 tccaacattt ctctatgtca tgcccatgtc gccaacaaga cttttttttg aggaaacctg 1 080 aaagatgcca tttggcctca tgcctttcga tctactgaag agaaaactaa tgfcacgatt 1140 gaagactctg ggtatccaag ttacaaaaat ttatgaagag gaatggtctt atattcctgt 1200 ttaccaaaca tgggggttct cagagcaaaa gaacctagca tttggtgctg cagcaagcat 1260 ggtgcatcca gcaacaggct attcggttgt acgatcacta tcagaagctc caaaatatgc 1320 ttctgtaaít gcaaagaíft tgaagcaaga taactctgca tatgtggttt ctggacaaag 1380 aacatttcaa cagtgcagta tgcaagcatg gagcagtctt tggccaaagg agcgaaaacg 1440 tcaaagagca ttctttcttt tcgggttaga gcttattgtg cagctagata ttgaagcaac 1500 cagaacgttc tttagaacct tcttccgctt gccaacttgg atgtggtggg gtttccttgg 1560 gtctt.cacta tcatctttcg atcttgtatt gttttccatg tacatgtttg ttttggcccc 1620 gaacagcatg aggatgtcac ttgtgagaca tttgctttca gatccttctg gtgcagttat 1680 ggttaaagct tacctcgaaa ggtaatctgt tttatgaaac tatagtgtct cattaaataa 1740 atgaggatcc ttcgtatatg tatatgatca tctctatgta tatcctatat tctaatctca 1800 aaaaaaaaaa aaaaaaaa taaagtaatc gaaaattcat tgatagaaaa 1848 <; 210 > 5 < 211 > 529 < 212 > PRT < 213 > Adonis palaestina < 400 > 5 Met Glu Leu Leu Gli Val Arg Asn Leu Be Ser Cis Pro Val Trp 1 5 10 15 Tre Fen Glí Tre Arg Asn Leu Ser Ser Lis Leu Aia Tir Asn lie 20 25 30 His Arg Tir Gli Ser Ser Cis Arg Val Asp Fen Gln Val Arg Ala Asp 35 40 45 Gli Gli Ser Gli Ser Arg Tre Ser Val Ala Tir Lis Glu Gli Fen Va. 50 55 60 Asp Glu Glu Asp Fen He Lis Wing Gli Gli Ser Glu Leu Leu Fen Val 65 70 75 80 Glp Met Gln Glp Tre Lis Ser Met Glu Lis Gln Ala Lis Leu Wing Asp 85 90 95 Lis Leu Pro Pro He Pro Fen Gli Glu Ser Val Met Asp Leu Val 100 105 110 He Gli Cis Gli Pro Wing Gli Leu Ser Leu Wing Wing Glu Wing Wing Us 115 120 125 Leu Gli Leu Lis Val Gli Leu lie Gli Pro Asp Leu Pro Fen Tre Asn 130 135 140 Asn Tir Gli Val Tf Glu Asp Glu Fen Lis Asp Leu Gli 1 Leu Glu Arg 145 150 155 160 Cis He Glu His Wing Tf Lis Asp Tre He Val Tir Leu Asp Asn Asp 165 170 175 Wing Pro Val Leu He Gli Arg Wing Tir Gli Arg Val Ser Arg His Leu 180 185 190 Leu His Glu Glu Leu Leu Lis Arg Cis Val Glu Ser Gli Val Ser Tir 195 200 205 Leu Asn Ser Lis Val Glu Arg He Tre Glu Wing Gli Asp Gli His Ser 210 215 220 Leu Val Val Cis Glu Asn Asp ee Fen He Pro Cis Arg Leu Wing Tre 225 230 235 240 Val Ala Ser Gli Ala Ala Ser Gli Lis Leu Leu Glu Tir Glu Val Gli 245 250 255 Gli Pro Arg Val Cis Val Gln Tre Ala Tir Gli Val Glu Val Glu Val 260 265 270 Glu Asn Asn Pro Tir Asp Pro Asn Leu Met Val Fen Met Asp Tir 275 280 285 Asp Tr Mef Gln Gln Us Leu Gin Cis Ser Glu Giu Tir Tir Tre 295 300 300 Fen Leu Tir Val Met Pro Met Ser Pro Tre Arg Leu Fen Fen Glu Glu 305 310 315 320 Tre Cis Leu Ala Ser Lis Asp Ala Met Pro Fen Asp Leu Leu Lis Arg 325 330 335 Lis Leu Met Ser Arg Leu Lis Tre Leu Gli He Gln Val Tre Lis He 340 345 350 Tir Glu Glu Glu Tf Ser Tir He Pro Val Gli Gli Ser Leu Pro Asn 355 360 365 Tre Glu Gln Lis Asn Leu Ala Fen Gli Ala Ala Ala Ser Met Val His 370 375 380 Pro Wing Tre Gli Tir Ser Val Val Arg Ser Leu Ser Glu Wing Pro Lis 385 390 395 400 Tir Aia Ser Val He Wing Lis Lie Leu Lis Gin Asp Asn Ser Wing Tir 405 410 415 Val Val Ser Gli Gln Ser Ser Wing Val Asn Lie Ser Met Gln Wing Tf 420 425 430 Ser Ser Leu Tf Pro Lis Glu Arg Lis Arg Gln Arg Wing Fen Fen Leu 435 440 445 Fen Gli Leu Glu Leu He Val Gln Leu Asp lie Glu Wing Tre Tre Arg Tre 450 455 460 Fen Fen Arg Tre Fen Fen Arg Leu Tre Tre Tf Met Tf Tf Gli Fen 465 470 475 480 Leu Gli Ser Ser Leu Ser Ser Fen Asp Leu Val Leu Fen Ser Met Tir 485 490 495 Met Fen Val Leu Ala Pro Asn Ser Met Arg Met Ser Leu Val Arg His 500 505 510 Leu Leu Ser Asp Pro Ser Gli Ala Val Met Val Lis Ala Tir Leu Glu 515 520 525 Arg < 210 > 6 < 211 > 530 < 212 > PRT < 213 > Adonis palaestina < 400 > 6 Met Glu Leu Leu Gli Val Arg Asn Leu He Ser Ser Cis Pro Val Tf 1 5 10 15 Tre Fen Gli Tre Arg Asn Leu Ser Ser Lis Leu Ala Tír Asn He 20 25 30 His Arg Tir Gli Ser Ser Cis Arg Val Asp Fen Gln Val Arg Wing Asp 35 40 45 Gli Gli Ser Gli Ser Arg Ser Ser Val AJa Tir Lis Glu Gli Fen Val 55 55 60 Asp Glu Glu Asp Fen He Lis Wing Gli Gli Ser Glu Leu Leu Fen Val 65 70 75 80 Gln Met Gln Gln Tre Lis Ser Met Glu Lis Gln Aia Lis Leu Ala Asp 85 90 95 Lis Pro Pro Pro Fep Gfi Ser Val Met Asp Leu Vai Val 100 105 110 He Gii Cis Gli Pro Ala Gli Leu Ser Leu Ala Ala Glu Ala Ala Lis 115 120 125 Leu Gli Leu Lis Val Gli Leu He Gli Pro Asp Leu Pro Fen Tre Asn 130 135 140 Asp Tír Gii Vaí Tf Glu Asp GIU Fep Lis Asp Leu Gíi Leu Glu Arg 145 150 155 160 Cis He Glu His Wing Tf Lis Asp Tre He Val Tir Leu Asp Asn Asp 165 170 175 Ata Pro Val Leu He Gli Arg Ala Tir Gli Arg Val Ser Arg His Leu 180 185 190 Leu His Glu Glu Leu Leu Lis Arg Cis Val Glu Ser Gli Val Ser Tir 195 200 205 Leu Asp Ser Lis Val Glu Arg He Tre Glu Glu Ala Asp Gli His Ser 210 215 220 Leu Val Val Cis Glu Asn Glu He Fen He Pro Cis Arg Leu Ala Tre 225 230 235 240 Val Ala Ser Gli Ala Ala Ser Gli Lis Leu Leu Glu Tir Glu Val Gli 245 250 255 Gli Pro Arg Val Cis Val Gln Tre Wing Tir Gli Val Glu Val Glu Val 260 265 270 Glu Asn Asn Pro Tir Asp Pro Asn Leu Met Val Fen Met Asp Tir Arg 275 280 285 Asp Tir Met Gln Gln Us Leu Gln Cis Ser Glu Glu Tir Pro Tre 290 295 300 Fen Leu Tir Val Met Pro Met Ser Pro Tre Arg Leu Fen Fen Glu Glu 305 310 315 320 Tre Cis Leu Ala Ser Lis Asp Aía Met Pro Fen Asp Leu Leu Lis Arg 325 330 335 Lis Leu Met Ser Arg Leu Lis Tre Leu Gli He Gln Va! Tre Lis Val 340 345 350 «36 Tir Glu Glu Glu Tf Ser Tir He Pro Val Gli Gli Ser Leu Pro Asn 355 360 365 Tre Giu Gln Lis Asn Leu Aia Fen Gli Aia Wing Wing Met Met Vai Hís 370 375 380 Pro Wing Tre Gli Tir Val Val Arg Ser Leu Ser Glu Ala Pro Lis 385 390 395 400 Tir Ala Ser Val He Ala Lis He Leu Lis Gln Asp Asn Ser Ala Tir 405 410 415 Val Val Ser Gli Gln Ser Ser Ala Val Asn He Ser Met Gln Ala Tf 420 425 430 Ser Ser Leu Trp Pro Lis Giu Arg Lis Arg Gln Arg Wing Fen Fen 435 440 445 Leu Fen Gli Leu Glu Leu He val Gln Leu Asp He Glu Wing Tre Arg 450 455 460 Tre Fen Fen Arg Tre Fen Fen Arg Leu Pro Tre Trp Met Tf Tf Gli 465 470 475 480 Fen Leu Gli Ser Ser Leu Ser Ser Fen Asp Leu Val Leu Fen Ser Met 485 490 495 Tir Met Fen Val Leu Ala Pro Asn Ser Met Arg Met Ser Leu Val Arg 500 505 510 His Leu Leu Ser Asp Pro Ser Gli Ala Val Met Val Arg Ala Tir Leu 515 520 525 Giu Arg 530

Claims (1)

1. CLAIMS 1. An isolated eukaryotic enzyme that converts lycopene to epsilon, epsilon-carotene. 2. An isolated eukaryotic enzyme of claim 1 having the amino acid sequence of SEQ ID NO: 2. 3. An isolated DNA sequence comprising a gene encoding the eukaryotic cyclase of claim 2. 4. The sequence of Isolated DNA according to claim 3, having the nucleic acid sequence of SEQ ID NO: 1. 5. An expression vector comprising the DNA sequence of claim 3. 6. A host containing the expression vector of claim 5. 7. The host of claim 6, wherein said host is E. coli. The host of claim 6, wherein said host is a plant. 9. The host of claim 8, wherein said host is calendula. The host of claim 8, wherein said host is tomato. eleven . A composition comprising the host of claim 6. 12. A composition comprising the host of claim 8. 13. A composition comprising bicyclic epsilon carotene obtained from the host of claim 6. 14. A composition comprising bicyclic epsilon carotene obtained from the host of claim 8. 15. A method of treating a disease comprising administering to a patient the same, an amount of the composition of claim 1 3 sufficient to treat said disease. 16. A method of treating disease comprising administering to a patient in need thereof, an amount of the composition of claim 14 sufficient to treat said disease.